Prebiotic proanthocyanidins inhibit bile reflux-induced esophageal adenocarcinoma through reshaping the gut microbiome and esophageal metabolome

Abstract

The gut and local esophageal microbiome progressively shift from healthy commensal bacteria to inflammatory-linked pathogenic bacteria in patients with gastroesophageal reflux disease, Barrett's esophagus and esophageal adenocarcinoma (EAC). However, mechanisms by which microbial communities and metabolites contribute to reflux-driven EAC remain incompletely understood and challenging to target. Herein, we utilized a rat reflux-induced EAC model to investigate targeting the gut microbiome-esophageal metabolome axis with cranberry proanthocyanidins (C-PAC) to inhibit EAC progression. Sprague Dawley rats, with or without reflux-induction received water or C-PAC ad libitum (700 μg/rat/day) for 25 or 40 weeks. C-PAC exerted prebiotic activity abrogating reflux-induced dysbiosis, and mitigating bile acid metabolism and transport, culminating in significant inhibition of EAC through TLR/NF-κB/P53 signaling cascades. At the species level, C-PAC mitigated reflux-induced pathogenic bacteria (Clostridium perfringens, Escherichia coli, and Proteus mirabilis). C-PAC specifically reversed reflux-induced bacterial, inflammatory and immune-implicated proteins and genes including Ccl4, Cd14, Crp, Cxcl1, Il6, Il1β, Lbp, Lcn2, Myd88, Nfkb1, Tlr2 and Tlr4 aligning with changes in human EAC progression, as confirmed through public databases. C-PAC is a safe promising dietary constituent that may be utilized alone or potentially as an adjuvant to current therapies to prevent EAC progression through ameliorating reflux-induced dysbiosis, inflammation and cellular damage.

Cranberry Proanthocyanidins Mitigate Reflux-Induced Transporter Dysregulation in an Esophageal Adenocarcinoma Model

“…We recently reported that C-PACs, rich in cranberry polyphenols, significantly inhibited EAC in a rat model through mitigating reflux-induced bacterial, inflammatory and immune-implicated proteins and genes as well as reducing esophageal bile acids, but through unknown processes [20]. These observations combined with metabolic enrichment results supported the hypothesis that C-PACs potentiate reflux-induced changes in bacterial metabolites, amino acids, fatty acids, bile acids and the TCA cycle, raising the question of transporter involvement [20]. In this study, we first comprehensively characterized bile reflux-induced transporter alterations in a rat EAC model and investigated C-PACs' capacity to mitigate reflux-induced transporter dysregulation.…”

“…A STRING protein interaction prediction was performed (Figure 2) using a list of 20 key transporters that are dysregulated in the same direction in both animal and human data, along with key proteins altered in the rat reflux-induced EAC model and mitigated by C-PACs, as previously described and evaluated herein (Figure 3) [20]. The protein interaction prediction revealed a complex connection within transporters and between other key proteins dysregulated or mutated in EAC.…”

“…To characterize transporter expression, we performed RNA sequencing and RT-PCR on esophageal RNA isolated from water-, C-PAC-and reflux-exposed rat esophagi with and without C-PAC treatment in the drinking water, as previously detailed [20]. Specifically, we utilized the Bio-Rad PrimePCR transporters plate, which is a predesigned assay containing optimized primers for 87 unique transporters and reference genes.…”

“…In this model, the duodenum is anastomosed to the gastroesophageal junction, leading to the reflux of bile and acidified gastric contents into the esophagus [21], mimicking GERD in humans. C-PACs inhibited esophageal EAC by 83% through reversing reflux-induced bacterial, inflammatory, and immune-implicated proteins and genes, with concomitant reductions in esophageal bile acids which drive EAC progression [20]. Herein, we extend these findings to (1) investigate whether C-PACs' mitigation of bile reflux-induced transporter dysregulation may mechanistically contribute to EAC inhibition in the rat reflux-induced EAC model and (2) characterize our preclinical results in the context of transporter dysregulation reported in human EAC, BE or normal squamous esophageal samples from non-cancer patients to inform the translational relevance of our findings.…”

“…Our lab recently reported that cranberry proanthocyanidins (C-PACs) inhibit esophageal adenocarcinoma progression in the rat esophagogastroduodenal anastomosis (EGDA) model of reflux-induced EAC [20]. In this model, the duodenum is anastomosed to the gastroesophageal junction, leading to the reflux of bile and acidified gastric contents into the esophagus [21], mimicking GERD in humans.…”